In situ hybridization of atrial natriuretic peptide mRNA in the endothelial cells of human umbilical vessels

The localization of mRNA encoding preproatrial natriuretic peptide (ANP) was investigated in cultured human umbilical vein endothelial cells (HUVEC) and tissue preparations of umbilical vein and artery. The techniques used were in situ hybridization and in situ hybridization combined with immunocytochemistry, using ³²P-radiolabelled and non-radioactive digoxigenin labelled complementary RNA probes. Human ANP mRNAs are mainly localized in the endothelial cells of the umbilical vein and, to a lesser extent, in the endothelial cells of the umbilical artery. The autoradiographic labelling and the intensity of digoxigenin staining were significantly reduced by treatment with RNase before in situ hybridization. This study provides unequivocal evidence for the expression of the ANP gene in the endothelial cells of human umbilical vessels, confirming that these endothelial cells have the ability to synthesize this peptide. The functional significance of the presence of the ANP mRNA in the endothelial cells of human umbilical vessels is discussed.     

Effect of natriuretic peptides on in vitro stimulated adrenocorticotropic hormone release and pro-opiomelanocortin mRNA expression by the fetal rat pituitary gland in late gestation

OBJECTIVE AND METHODS: We investigated the effects of individual natriuretic peptides (atrial natriuretic peptide, ANP; brain natriuretic peptide, BNP, and C-type natriuretic peptide, CNP) on rat corticotropin-releasing factor stimulated adrenocorticotropic hormone (ACTH) secretion by the pituitary gland of 21-day-old rat fetuses in vitro and on pro-opiomelanocortin gene expression using in situ hybridization. RESULTS: Graded concentrations of ANP, BNP, or CNP (10(-10), 10(-9), and 10(-8) mol/l) induced a log dose dependent inhibition of ACTH secretion induced by rat corticotropin-releasing factor (10(-10) mol/l). These natriuretic peptides showed equipotent effects on a molar basis. Moreover, ANP, BNP, or CNP at 10(-10) mol/l reduced significantly the pituitary pro-opiomelanocortin mRNA expression. In addition, the immunoreactive ANP, BNP, and CNP cells were localized in the anterior lobe, but not in the intermediate lobe of the fetal pituitary gland. CONCLUSIONS: These data suggest that the fetal pituitary gland may be both a source and a target for natriuretic peptides that might control ACTH synthesis and release via an endocrine and/or paracrine mechanism. The natriuretic peptides could participate, as well as glucocorticoids, in the control of the corticotropin-stimulating activity of the fetal rat in late gestation.     

肾上腺髓质素在正常大鼠脑中的分布-免疫组化及原位杂交分析

目的验证大鼠脑内是否存在ADM及其mRNA。方法取10只健康雄性SD大鼠,体重200-250g,应用免疫组织化学法和原位杂交组织化学法检测正常大鼠脑内ADM及其mRNA的表达情况。结果在大鼠脑内ADM及其mRNA阳性细胞主要表达在大脑皮质、海马结构、齿状回、丘脑、室旁组织、脉络丛、室管膜细胞、基底节、血管内皮细胞,其中脉络丛、室旁组织、丘脑为高表达区,其次为大脑皮质、海马。在大鼠大脑内ADMmRNA的表达与ADM阳性细胞的表达相一致。结论ADM及其mRNA在大脑内广泛表达,提示ADM在中枢神经系统内具有重要的作用。     

肾上腺髓质素在正常大鼠脑中的分布一免疫组化及原位杂交分析

目的验证大鼠脑内是否存在ADM及其mRNA。方法取10只健康雄性SD大鼠,体重200-250g,应用免疫组织化学法和原位杂交组织化学法检测正常大鼠脑内ADM及其mRNA的表达情况。结果在大鼠脑内ADM及其mRNA阳性细胞主要表达在大脑皮质、海马结构、齿状回、丘脑、室旁组织、脉络丛、室管膜细胞、基底节、血管内皮细胞,其中脉络丛、室旁组织、丘脑为高表达区,其次为大脑皮质、海马。在大鼠大脑内ADMmRNA的表达与ADM阳性细胞的表达相一致。结论ADM及其mRNA在大脑内广泛表达,提示ADM在中枢神经系统内具有重要的作用。     

Ultrastructural characterization of atrial natriuretic peptide receptors (ANP-R) mRNA expression in rat kidney cortex

Atrial natriuretic peptide (ANP) and two complementary peptides named brain natriuretic peptide and C-type natriuretic peptide are involved in diuresis, natriuresis, hypotension and vasorelaxation. Their actions are mediated by highly selective and specific ANP receptors. Three subtypes have been characterized and cloned: ANP receptor A, ?B and ?C. In the present study, the mRNA for each subtype was detected by ultrastructural in situ hybridization on ultrathin sections of Lowicryl-embedded tissue and frozen tissue. The distribution of mRNA (visualized by gold particles) for each subtype was found to differ in different cells of the nephron. The three subtypes of this receptor family were expressed in all the parts of the nephron, but their expression levels were different. The ANPR-A mRNA was the most abundant in cells of glomerulus, proximal and distal tubules. The subtype C was the least expressed mRNA in glomerulus. In contrast, the subcellular localization of the three mRNAs was similar; they were found in the cytoplasmic matrix and the euchromatin of the nucleus. In conclusion, the differential expression of these mRNAs in kidney cortex indicates that these three peptides act directly in differing parts of nephron regions which are the glomerulus, the proximal and distal tubules.     

Relationship between arginine vasotocin-like and natriuretic peptide-like immunoreactive structures in the brain of the toad Bufo marinus

The distribution of natriuretic peptide-like immunoreactivity was investigated in the brain of Bufo marinus and compared with arginine vasotocin-like immunoreactivity using fluorescence immunohistochemistry. The antisera used were rabbit anti-porcine brain natriuretic peptide, which recognises the three main structural forms of natriuretic peptides, and guinea-pig antivasopressin, which recognises arginine vasotocin. Natriuretic peptide-like immunoreactive fibres were observed in many regions of the brain, being densest in the preoptic/hypothalamic region of the diencephalon and the interpeduncular nucleus of the mesencephalon. Natriuretic peptide-like immunoreactive cell bodies were observed in the dorsal and medial pallium, the medial amygdala, the preoptic nucleus, the ventral hypothalamus, the nucleus posterodorsalis tegmenti mesencephali, and the interpeduncular nucleus. No natriuretic peptide-like immunoreactivity was seen in the pituitary gland. The distribution of arginine vasotocin-like immunoreactivity was similar to that described previously for other amphibian species. Numerous immunoreactive cell bodies were present in the preoptic nucleus whilst immunoreactive fibres were observed in the preoptic/hypothalamic region as well as in extrahypothalamic regions such as the medial amygdala and the medial pallium. Double-labelling immunohistochemistry revealed no colocalisation of arginine vasotocin-like and natriuretic peptide-like immunoreactivities in the same neural elements. The results suggest that natriuretic peptides and arginine vasotocin have distinct distributions in the brain but that natriuretic peptide-like immunoreactive fibres in the hypothalamus could influence the activity of arginine vasotocin-like immunoreactive cell bodies.     

A synthetic oligonucleotide probe encoding for atrial natriuretic peptide detects specific mRNA transcripts in rat heart but not brain using in situ hybridization histochemistry

In situ hybridization histochemical techniques were used in an attempt to demonstrate atrial natriuretic peptide (ANP) messenger RNA (mRNA) in the rat brain. A synthetic oligonucleotide derived from previously reported ANF cDNA sequence was used as a probe. Northern blot analysis of total RNA isolated from rat heart demonstrated that the oligonucleotide recognized a single species of RNA (0.9 kb), a size consistent with previous reports. Rat heart sections revealed dense accumulations of ANF mRNA in the cardiac atria and lesser densities in the ventricles. Rat brain sections hybridized with the same oligonucleotide did not label ANF mRNA accumulations in any neuronal cell bodies. A possible explanation for this latter observation is either sparsely distributed expressing neurons or low expression and high turnover of ANF mRNA in brain.     

2Hz和100Hz电针加速脑内三种阿片肽基因表达

我室以往的工作证明２Ｈｚ和１００Ｈｚ电针可引起中枢释放不同种类的阿片肽,本工作试图阐明不同频率的电针是否影响三种阿片肽的基因转录。用地高辛标记的反义ｃＲＮＡ探针进行原位杂交,显示大鼠脑内前脑啡肽原（ＰＰＥ）,前强啡肽强（ＰＰＤ）和前阿黑皮素原（ＰＯＭＣ）ｍＲＮＡ。结果：（１）低、高频电针均不影响ＰＯＭＣ ｍＲＮＡ的水平。（２）对ＰＰＥ的影响,两种频率电针诱导脑干网状结构头端腹内侧区ＰＰＥ ｍＲＮＡ     

Regional expression of a MCD-peptide and dendrotoxin I-sensitive voltage-dependent potassium channel in rat brain

In situ hybridization histochemistry has been used to analyze the regional expression of a class of voltage-dependent K+ channel that is sensitive to two polypeptide toxins (MCD peptide and dendrotoxin I) that produce spectacular effects on brain function. A heterogeneous expression of this K+ channel was observed throughout the brain. High mRNA contents were observed in the granule cells of the gyrus dentatus as well as in pyramidal cells of the Ammon horn (CA3 greater than CA1) and in the cerebellum. Conversely, low levels of expression were found in basal ganglia (caudate putamen, globus pallidus, and ventral pallidum).     

Distribution of Mahogany/Attractin mRNA in the rat central nervous system

The Mahogany/Attractin gene (Atrn) has been proposed as a downstream mediator of Agouti signaling because yellow hair color and obesity in lethal yellow (A(y)) mice are suppressed by the mahogany (Atrn(mg)) mutation. The present study examined the distribution of Atrn mRNA in the brain and spinal cord by in situ hybridization. Atrn mRNA was found widely distributed throughout the central nervous system, with high levels in regions of the olfactory system, some limbic structures, regions of the brainstem, cerebellum and spinal cord. In the hypothalamus, Atrn mRNA was found in specific nuclei including the suprachiasmatic nucleus, the supraoptic nucleus, the medial preoptic nucleus, the paraventricular hypothalamic nucleus, the ventromedial hypothalamic nucleus, and the arcuate nucleus. These results suggest a broad spectrum of physiological functions for the Atrn gene product.     

阿片受体样受体ORL1基因在大鼠脑内的表达

我们采用地主辛标记的寡聚核苷酸探针和原位杂产技术研究了新发现的阿片受体样受体ＯＲＬ１基因在正常大鼠脑仙的表达和分布。发现ＯＲＬ１基因在大鼠脑内广泛表达。尤其在大脑皮层、丘脑、下丘脑、杏仁核、海马、隔核、缰核、导水管周围灰质、中缝核群及蓝斑等区域。提示ＯＲＬ１除参与痛觉调制外,还可能参与脑内多项生理功能的调控。     

Atrial natriuretic peptide-like immunoreactivity in astrocytes of parenchyma and glia limitans of the canine brain.

The Na(+)-regulating and vasoactive peptide hormone atrial natriuretic peptide (ANP) has been localized in cultured glial cells from neonatal rat brain but has not been reported in situ in adult brain. In the present study, immunohistochemical localization of ANP in astrocytes of adult canine brain was obtained using an antibody against rat ANF IV, a 25-amino acid fragment identical to low molecular weight brain ANP. ANP-like immunoreactivity was localized in widely dispersed astroglia throughout the canine brain, including Types I and II, in addition to some astrocytes of the glia limitans (GL).     

Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family

Cells expressing mRNA for hippocampus-derived neurotrophic factor (HDNF/NT-3) or brain-derived neurotrophic factor (BDNF) were identified by in situ hybridization. In the rat brain, HDNF mRNA was predominantly found in pyramidal neurons in CA1 and CA2 of the hippocampus. Lower levels of HDNF mRNA were found in granular neurons of the dentate gyrus and in neurons of the taenia tecta and induseum griseum. BDNF mRNA-expressing cells were more widely distributed in the rat brain, with high levels in neurons of CA2, CA3, and the hilar region of the dentate gyrus, in the external and internal pyramidal layers of the cerebral cortex, in the claustrum, and in one brainstem structure. Lower levels were seen in CA1 and in the granular layer of the hippocampus, in the taenia tecta, and in the mammillary complex. In peripheral tissues, HDNF mRNA was found in glomerular cells in the kidney, secretory cells in the male rat submandibular gland, and epithelial cells in secondary and tertiary follicles in the ovary. Cells expressing BDNF mRNA were found in the dorsal root ganglia, where neurons of various sizes were labeled.     

大鼠基底核心房肽mRNA的表达——原位杂交研究

采用原位杂交技术,研究大鼠基底核团细胞内是否存在ANPmRNA的基因表达,结果发现在尾壳核细胞内存在阳性反应产物,而苍白珠细胞内示见阳性反应。说明在尾壳核内存在ANP的基因表达及ANP的合成。实验结果为尾壳核产生分泌ANP提供了形态学依据。